scholarly journals Delivery of Metabolically Neuroactive Probiotics to the Human Gut

2021 ◽  
Vol 22 (17) ◽  
pp. 9122
Author(s):  
Peter A. Bron ◽  
Marta Catalayud ◽  
Massimo Marzorati ◽  
Marco Pane ◽  
Ece Kartal ◽  
...  
Keyword(s):  
De Novo ◽  
Ex Vivo ◽  
Human Microbiome ◽  
Live Cells ◽  
Metabolomics Data ◽  
Strain Characterization ◽  
Probiotic Product ◽  
Microbial Strains ◽  
Microbial Strain

The human microbiome is a rich factory for metabolite production and emerging data has led to the concept that orally administered microbial strains can synthesize metabolites with neuroactive potential. Recent research from ex vivo and murine models suggests translational potential for microbes to regulate anxiety and depression through the gut-brain axis. However, so far, less emphasis has been placed on the selection of specific microbial strains known to produce the required key metabolites and the formulation in which microbial compositions are delivered to the gut. Here, we describe a double-capsule technology to deliver high numbers of metabolically active cells derived from the 24-strain probiotic product SH-DS01 to the gastrointestinal tract, including the small intestine, where immune responses and adsorption of metabolites into the bloodstream occur. Based on its genome sequence, Limosilactobacillus reuteri SD-LRE2-IT was predicted to have the genetic capacity to de novo produce a specific metabolite of interest to brain health, vitamin B12, which could be confirmed in vitro. Taken together, our data conceptualizes the importance of rationally defined microbial strain characterization based on genomics and metabolomics data, combined with carefully designed capsule technology for delivery of live cells and concomitant functionality in and beyond the gut ecosystem.

Abstract 11607: Novel Assay for Detection of LDL Transcytosis Across Coronary Endothelium Reveals an Unexpected Role for SR-B1

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Susan M Armstrong ◽  
Michael G Sugiyama ◽  
Andrew Levy ◽  
Dante Neculai ◽  
Mark Roufaiel ◽  
...  
Keyword(s):  
Coronary Artery ◽  
Fluorescence Microscopy ◽  
Ex Vivo ◽  
Scavenger Receptor ◽  
Intercellular Junctions ◽  
Live Cells ◽  
Human Coronary Artery ◽  
Class B ◽  
Major Route

Introduction: Retention of LDL beneath the arterial endothelium initiates an inflammatory response culminating in atherosclerosis. How LDL crosses the endothelium to enter the arterial wall remains unknown. While LDL could conceivably pass between endothelial cells (paracellularly) or through them (transcytosis), electron microscopy studies in animals revealed LDL in intracellular vesicles and none at intercellular junctions. This, combined with the absence of endothelial injury or intercellular gaps in early atherosclerosis, suggests that transcytosis is the major route. However, technical challenges with studying transcytosis have made confirming and extending these findings difficult. We developed and validated a novel assay for measuring the transcytosis of native LDL across live human coronary artery endothelium in vitro. Using this assay, we propose to elucidate the regulation of LDL transcytosis and have identified a novel role for SR-B1. Methods and Results: Experiments were performed using primary human coronary artery endothelial monolayers. Transcytosis was quantified in single live cells in real time using total internal reflectance fluorescence microscopy. Transcytosis of LDL was saturable and inhibited by excess unlabeled LDL. By fluorescence microscopy we found that DiI-LDL colocalized significantly with scavenger receptor, class B, type 1 (SR-B1). Unexpectedly, overexpression of SR-BI resulted in increased LDL transcytosis, while knockdown of SR-BI by siRNA inhibited transcytosis. Excess HDL, the canonical SR-B1 ligand, also decreased LDL transcytosis. To confirm the occurrence of transcytosis in an intact vessel, we perfused murine aortas ex vivo with both LDL and dextran of a smaller molecular radius. We observed the accumulation of subendothelial LDL without dextran, indicating that transcytosis of LDL occurs in intact vessels. Conclusions: The accumulation of LDL in the subendothelial intima is the first step of atherosclerosis yet little is known about how it occurs. Our data suggests that transcytosis of LDL is an important contributor, particularly in the early stages of the disease. By identifying the mechanisms of transcytosis, our work could have important implications for its pathogenesis and therapy.

Correlation guided Network Integration (CoNI) reveals novel genetic regulators of hepatic metabolism

2020 ◽  
Author(s):  
Valentina S. Klaus ◽  
Sonja C. Schriever ◽  
Andreas Peter ◽  
José Manuel Monroy Kuhn ◽  
Martin Irmler ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Hepatic Metabolism ◽  
Omics Data ◽  
Network Integration ◽  
Data Set ◽  
Metabolomics Data ◽  
Different Types ◽  
Hepatic Fat ◽  
Liver Biopsies

ABSTRACTThe steadily increasing amount of newly generated omics data of various types from genomics to metabolomics is a chance and a challenge to systems biology. To fully use its potential, one key is the meaningful integration of different types of omics. We here present a fully unsupervised and versatile correlation-based method, termed Correlation guided Network Integration (CoNI), to integrate multi-omics data into a hypergraph structure that allows for identification of effective regulators. Our approach further unravels single transcripts mapped to specific densely connected metabolic sub-graphs or pathways. By applying our method on transcriptomics and metabolomics data from murine livers under standard chow or high-fat-diet, we isolated eleven genes with a regulatory effect on hepatic metabolism. Subsequent in vitro and ex vivo experiments in human liver cells and human obtained liver biopsies validated seven candidates including INHBE and COBLL1, to alter lipid metabolism and to correlate with diabetes related traits such as overweight, hepatic fat content and insulin resistance (HOMA-IR). Last, we successfully applied our methods to an independent data-set to confirm its versatile and transferable character.

Sensitivity of Childhood Acute Lymphoblastic Leukemia (ALL) to Idarubicin Is Significantly Higher Than to Daunorubicin Treatment Based on Ex Vivo Apoptosis Induction.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4495-4495
Author(s):  
Aram Prokop ◽  
Banu Bagci ◽  
Guenaelle Lingfeld ◽  
Lucia Badiali ◽  
Karin Garbrecht ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Response Rate ◽  
De Novo ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Cell Populations ◽  
Apoptosis Induction ◽  
Good Tolerability ◽  
Childhood All

Abstract Anthracyclines, especially daunorubicin, play a very important role in the treatment of acute lymphoblastic leukemia (ALL) and the relapsed ALL in childhood. In the present study, primary lymphoblasts isolated from 65 children with de novo ALL (median: 5.8 years; range: 1.9 – 16.9 years) and relapsed ALL (median: 12.7 years; range: 1.3 – 17.9 years) were treated with daunorubicin (10 mmol/l) or idarubicin (2 mmol/l) in vitro. We could show that both anthracylines induce apoptosis, as evidenced by measurement of genomic DNA fragmentation. Interestingly, daunorubicin only induced modest apoptosis, whereas idarubicin displayed a significantly stronger apoptosis inducing effect. Furthermore the treatment of daunorubicin-resistant lymphoblasts with idarubicin resulted in good response in most of the resistant cell populations. Out of the 65 patients analysed in this study 23 were female (13 de novo ALL, 10 relapsed ALL) and 42 were male (29 de novo ALL, 13 relapsed ALL). Primary lymphoblasts were obtained by bone marrow aspiration and separated by centrifugation over Ficoll. Within these cell populations following immunologic subgroups were found: 35 c-ALL, 10 pre-B-ALL, 7 pro-B-ALL, 10 T-ALL and 3 pre-T-ALL. Daunorubicin induced apoptosis in 33 out of 65 lymphoblast populations (response rate 50.8 %). Nevertheless, a far higher response rate was observed for idarubicin with 59/65 (90,8 %) (p < 0.008), if response is defined as apoptosis induction higher than 1 %. Daunorubicin-resistance was found in 32/65 (49,2 %), resistance to both was observed in 6/65 (9,2 %). Treatment of daunorubicin-resistant lymphoblasts with idarubicin resulted in significant apoptosis induction in 26 out of 32 cell populations (81,3 %). We clearly demonstrated here that the in vitro treatment of lymphoblasts from children with de novo or relapsed ALL with idarubicin induces significantly higher response rates than daunorubicin treatment. The ex vivo sensitivity of daunorubicin-resistant lymphoblasts of childhood ALL to idarubicin treatment reflects the better potency of idarubicin to induce apoptosis and to overcome daunorubicin resistance. These data prompted us to study the clinical relevance of idarubicin in ongoing clinical trials to improve existing therapeutic regiments. First clinical data point to a good tolerability of idarubicin in the treatment of relapsed ALL in childhood.

scholarly journals Signatures of immune reprogramming in anti-CD52 therapy of MS: markers for risk stratification and treatment response

2019 ◽  
Vol 1 (1) ◽  
Author(s):  
Laura Bierhansl ◽  
Tobias Ruck ◽  
Steffen Pfeuffer ◽  
Catharina C. Gross ◽  
Heinz Wiendl ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Adverse Events ◽  
Treatment Response ◽  
De Novo ◽  
Ex Vivo ◽  
Phase Iii ◽  
Serum Samples ◽  
Two Phase ◽  
Relapsing Remitting

Abstract Background Multiple sclerosis is one of the most prevalent neurological diseases in young adults affecting over 2 million people worldwide. Alemtuzumab is a highly effective therapy in relapsing remitting MS. Alemtuzumab is a monoclonal CD52 antibody that proved its efficacy against an active comparator (interferon [IFN]-β1a) in a phase II trial and two phase III trials regarding clinical and MRI outcomes. Nevertheless, the exact mode of action is still unknown. Alemtuzumab is commonly associated with secondary autoimmune disorders significantly affecting the risk-benefit ratio. Therefore, new biomarkers predicting treatment response and adverse events are urgently needed. This study aims to further elucidate the mechanism of action of the neuroprotective potential of alemtuzumab in relapsing-remitting multiple sclerosis (RRMS). Methods/Design This is a 3-year multicentre, explorative study including overall 150 patients comprising three different groups: (i) de novo patients prior and after alemtuzumab treatment initiation, (ii) patients under alemtuzumab treatment and (iii) patients requiring more than two alemtuzumab infusions. Peripheral blood and serum samples will be collected semi-annually for several in vitro/ex vivo assays to detect and characterize immune cells including their functional activity. Furthermore, data of MRI scans and disease-related impairment (using EDSS and MSFC), as well as the number and time of relapses, will be assessed. The clinical study is registered at clinicaltrials.gov (NCT04082260). Perspective Our study will provide deep insights into the underlying immunological changes in a longitudinal analysis of alemtuzumab treated RRMS patients. By combining clinical, radiological and functional immune-phenotype data, we will be able to identify biomarkers and/or immune signatures predicting treatment response and adverse events. Thereby, the understanding of the mechanisms of action of alemtuzumab will improve its efficacy and safety for present and future patients.

Melphalan and XPO1 Inhibitor Combination Therapy for the Treatment of Multiple Myeloma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2084-2084 ◽  
Author(s):  
Joel G Turner ◽  
Jana L Dawson ◽  
Steven Grant ◽  
Kenneth H. Shain ◽  
Yun Dai ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Lines ◽  
De Novo ◽  
Ex Vivo ◽  
Myeloma Cell ◽  
Drug Resistant ◽  
Cell Viability Assay ◽  
Myeloma Cells ◽  
Multiple Myeloma Cell

Abstract Introduction High-dose melphalan chemotherapy with autologous stem cell transplant remains the standard of care for the treatment of multiple myeloma. However, patients eventually develop drug resistance and die from progressive disease despite the introduction of therapies using proteosome inhibitors (PIs) and immunomodulatory drugs (IMIDs). The incurable nature of multiple myeloma clearly demonstrates the need for novel agents and treatments. Here, our aim was to investigate whether the use of XPO1 (exportin 1, CRM1) inhibitors (XPO1i) could sensitize de novo and acquired drug-resistant multiple myeloma cells both in vitro and ex vivo to the alkylating agent melphalan. Materials and Methods Human multiple myeloma cell lines NCI-H929, RPMI-8226, U266 and PBMC controls were treated in vitro with the XPO1i KOS-2464 and the orally available Selective Inhibitor of Nuclear Export (SINE) selinexor (KPT-330) or) +/- melphalan. Multiple myeloma cells were grown at high-density conditions (>3-5x106 cells/mL). High-density multiple myeloma cells have been shown to possess de novo drug resistance. Sensitivity of the XPO1i/melphalan-treated NCI-H929 cells was measured by cell viability assay (CellTiter-Blue). Apoptosis in XPO1i/melphalan-treated NCI-H929, RPMI-8226, and U266 cells was assayed using flow cytometry (activated caspase 3). Proximity ligation assays were performed to assess XPO1-p53 binding in the presence of an XPO1i. Western blots of XPO1i-treated myeloma cells were performed for nuclear and total p53. Drug-resistant U266 (PSR) and 8226 (8226/B25) myeloma cell lines were developed by incremental exposure to bortezomib. PSR cells are able to grow in 15 nM bortezomib and the 8226/B25 in 25 nM. These resistant myeloma cells were treated in vitro with XPO1i +/- melphalan. Sensitivity to therapy was measured by apoptosis and cell viability assay. Multiple myeloma cells isolated from patients with newly diagnosed, relapsed, or refractory disease were treated with XPO1i +/- melphalan and CD138+/light chain+ myeloma cells and assayed for apoptosis. Results Multiple myeloma cell (NCI-H929) viability was decreased synergistically by XPO1i when used in combination with melphalan, as shown by the calculated combinatorial index (CI) values. We examined sequencing of the drugs and found that concurrent treatment with melphalan (10 µM) and selinexor (300 nM) for 48 hours produced the best results (CI value 0.370, n=6). Sequential treatment (selinexor for 24 hours followed by melphalan for an additional 24 hours) or the reverse sequence had slightly less synergy, with CI values of 0.491 (n=9) and 0.565 (n=3), respectively. Normal PBMC control cells were unaffected by XPO1i/melphalan treatment as shown by viability and apoptotic assays. Proximity ligation assay demonstrated that XPO1i blocks XPO1/p53 binding. Western blot showed that the XPO1i treatment of myeloma cells increased nuclear and total p53. Drug-resistant 8226/B25 myeloma cells but not PSR cells were found to be resistant to melphalan when compared to parental cell lines. Both resistant myeloma cell lines were sensitized by XPO1i to melphalan as shown by apoptosis assay (3- to 10-fold). CD138+/light chain+ myeloma cells derived from newly diagnosed, relapsed, and refractory myeloma patients were also sensitized by XPO1 inhibitors to melphalan as demonstrated by apoptotic assays (e.g. activated caspase 3). Conclusions XPO1i synergistically improved the response of de novo and acquired drug-resistant myeloma cells to melphalan in vitro and ex vivo. It is possible that this synergy may be due to an increase of nuclear p53 by XPO1i and the reported activation of p53 by melphalan. Future studies include in vitro experiments using drug-resistant human U266 myeloma cells in NOD-SCID-gamma mice and clinical trials using melphalan in combination with the SINE selinexor. Combination therapies using selinexor and melphalan may significantly improve the treatment of myeloma. Disclosures Kauffman: Karyopharm Therapeutics: Employment. Shacham:Karyopharm Therapeutics: Employment.

scholarly journals Integrin α4β7 Is Downregulated on the Surfaces of Simian Immunodeficiency Virus SIVmac239-Infected Cells

2010 ◽  
Vol 84 (13) ◽  
pp. 6344-6351 ◽  
Author(s):  
Melisa L. Budde ◽  
Jennifer J. Lhost ◽  
Dawn M. Dudley ◽  
Eva G. Rakasz ◽  
David H. O'Connor
Keyword(s):  
Simian Immunodeficiency Virus ◽  
Immune Evasion ◽  
Lymphoid Tissue ◽  
De Novo ◽  
Ex Vivo ◽  
Viral Proteins ◽  
Flow Cytometry Assay ◽  
Immunodeficiency Virus ◽  
Infected Cells

ABSTRACT Simian immunodeficiency virus (SIV) and human immunodeficiency virus (HIV) infection results in an early and enduring depletion of intestinal CD4+ T cells. SIV and HIV bind integrin α4β7, thereby facilitating infection of lymphocytes that home to the gut-associated lymphoid tissue (GALT). Using an ex vivo flow cytometry assay, we found that SIVmac239-infected cells expressed significantly lower levels of integrin α4β7 than did uninfected cells. This finding suggested a potential viral effect on integrin α4β7 expression. Using an in vitro model, we confirmed that integrin α4β7 was downregulated on the surfaces of SIVmac239-infected cells. Further, modulation of integrin α4β7 was dependent on de novo synthesis of viral proteins, but neither cell death, the release of a soluble factor, nor a change in activation state was involved. Downregulation of integrin α4β7 may have an unappreciated role in the CD4 depletion of the mucosal-associated lymphoid compartments, susceptibility to superinfection, and/or immune evasion.

scholarly journals Discovery of tumoricidal DNA oligonucleotides by effect-directedin-vitroevolution

2019 ◽  
Author(s):  
Noam Mamet ◽  
Yaniv Amir ◽  
Erez Lavi ◽  
Liron Bassali ◽  
Gil Harari ◽  
...  
Keyword(s):  
Drug Discovery ◽  
De Novo ◽  
Ex Vivo ◽  
Binding Capacity ◽  
Biological Effects ◽  
Current Model ◽  
Target Cells ◽  
Dna Oligonucleotides

AbstractOur current model of drug discovery is challenged by the relative ineffectiveness of drugs against highly variable and rapidly evolving diseases and their relatively high incidence of adverse effects due to poor selectivity. Here we describe a robust and reproducible platform which could potentially address these limitations. The platform enables rapid,de-novodiscovery of DNA oligonucleotides evolvedin-vitroto exert specific biological effects on target cells. Unlike aptamers, which are selected by their ligand binding capacity, this platform is driven directly by therapeutic effect and selectivity towards target vs negative target cells. The process could, therefore, operate without anya-prioriknowledge (e.g. mutations, biomarker expression, or known drug resistance) of the target. We report the discovery of DNA oligonucleotides with direct and selective cytotoxicity towards several tumor cell lines as well as primary, patient-derived solid and hematological tumors, some with chemotherapy resistance. Oligonucleotides discovered by this platform exhibited favorable biodistribution in animals, persistence in target tumors up to 48 hours after injection, and safety in human blood. These oligonucleotides showed remarkable efficacyin-vivoas well asex-vivoin freshly obtained, 3D cultured human tumors resistant to multiple chemotherapies. With further improvement, these findings could lead to a drug discovery model which is target-tailored, mechanism-flexible, and nearly on-demand.

TPEN Selectively Eliminates Lymphoblastic B cells from Bone Marrow Pediatric Acute Lymphoblastic Leukemia Patients

2021 ◽  
Author(s):  
Miguel Mendivil-Perez ◽  
Carlos Velez-Pardo ◽  
Lina Maria Quiroz-Duque ◽  
Alexandra Restrepo-Rincon ◽  
Natalia Andrea Valencia-Zuluaga ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Lymphoblastic Leukemia ◽  
Anticancer Agents ◽  
Pediatric Patients ◽  
De Novo ◽  
Ex Vivo ◽  
Lymphoblastic Leukemia ◽  
Leukemic Cell ◽  
Leukemia Cells

B-cell acute lymphoblastic leukemia (B-ALL) is a hematologic disorder characterized by the abnormal proliferation and accumulation of immature B-lymphoblasts arrested at various stages of differentiation. Despite advances in treatment, a significant percentage of pediatric patients with precursor B-ALL still relapse. Therefore, alternative therapies are needed to improve the cure rates for pediatric patients. TPEN (N, N, N&rsquo;, N&rsquo;-tetrakis(2-pyridylmethyl)-ethylenediamine).is a pro-oxidant agent capable of selectively inducing apoptosis in leukemia cells. Consequently, it has been suggested that TPEN could be a potential agent for oxidative therapy. However, it is not yet known whether TPEN can selectively destroy leukemia cells in a more disease-like model, for example, the bloodstream and bone marrow (BM), in vitro. This investigation is an extension of a previous study that dealt with the effect of TPEN on ex vivo isolated/purified refractory B-ALL cells. Here, we evaluated the effect of TPEN on whole BM from nonleukemic patients (control) or pediatric patients diagnosed with de novo B-ALL or refractory B-ALL cells by analyzing the hematopoietic cell lineage marker CD34/CD19. Although TPEN was innocuous to nonleukemic BM (n=3), we found that TPEN significantly induced apoptosis in de novo (n = 5) and refractory B-ALL (n = 6) leukemic cell populations. Moreover, TPEN significantly increased the counts of cells positive for the oxidation of the stress sensor protein DJ-1, a sign of the formation of H2O2, and significantly increased the counts of cells positive for the pro-apoptotic proteins TP53, PUMA, and CASPASE-3 (CASP-3), indicative of apoptosis, in B-ALL cells. We demonstrate that TPEN selectively eliminates B-ALL cells independent of age, diagnosis status (de novo or refractory), sex, karyotype, or immunophenotype. Understanding TPEN-induced cell death in leukemia cells provides insight into more effective therapeutic oxidation-inducing anticancer agents.

scholarly journals Sialic acid-modified antigens impose tolerance via inhibition of T-cell proliferation and de novo induction of regulatory T cells

2016 ◽  
Vol 113 (12) ◽  
pp. 3329-3334 ◽  
Author(s):  
Maurizio Perdicchio ◽  
Juan M. Ilarregui ◽  
Marleen I. Verstege ◽  
Lenneke A. M. Cornelissen ◽  
Sjoerd T. T. Schetters ◽  
...  
Keyword(s):  
T Cells ◽  
Sialic Acid ◽  
T Cell ◽  
De Novo ◽  
Ex Vivo ◽  
Treg Cells ◽  
Sialic Acids ◽  
Antigen Uptake

Sialic acids are negatively charged nine-carbon carboxylated monosaccharides that often cap glycans on glycosylated proteins and lipids. Because of their strategic location at the cell surface, sialic acids contribute to interactions that are critical for immune homeostasis via interactions with sialic acid-binding Ig-type lectins (siglecs). In particular, these interactions may be of importance in cases where sialic acids may be overexpressed, such as on certain pathogens and tumors. We now demonstrate that modification of antigens with sialic acids (Sia-antigens) regulates the generation of antigen-specific regulatory T (Treg) cells via dendritic cells (DCs). Additionally, DCs that take up Sia-antigen prevent formation of effector CD4+ and CD8+ T cells. Importantly, the regulatory properties endowed on DCs upon Sia-antigen uptake are antigen-specific: only T cells responsive to the sialylated antigen become tolerized. In vivo, injection of Sia-antigen–loaded DCs increased de novo Treg-cell numbers and dampened effector T-cell expansion and IFN-γ production. The dual tolerogenic features that Sia-antigen imposed on DCs are Siglec-E–mediated and maintained under inflammatory conditions. Moreover, loading DCs with Sia-antigens not only inhibited the function of in vitro–established Th1 and Th17 effector T cells but also significantly dampened ex vivo myelin-reactive T cells, present in the circulation of mice with experimental autoimmune encephalomyelitis. These data indicate that sialic acid-modified antigens instruct DCs in an antigen-specific tolerogenic programming, enhancing Treg cells and reducing the generation and propagation of inflammatory T cells. Our data suggest that sialylation of antigens provides an attractive way to induce antigen-specific immune tolerance.

Prognostic Impact of Combined Ex Vivo Drug Resistance to Fludarabine, Treosulphan and Mitoxantrone in Childhood Acute Myeloid Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2780-2780
Author(s):  
Jan Styczynski ◽  
Mariusz Wysocki ◽  
Agnieszka Dluzniewska ◽  
Krzysztof Czyzewski ◽  
Robert Debski ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Prognostic Value ◽  
De Novo ◽  
Ex Vivo ◽  
Prognostic Significance ◽  
Resistance Profile ◽  
Drug Resistance Profile ◽  
Childhood Acute Myeloid Leukemia ◽  
Acute Myeloid

Abstract Combined ex vivo drug resistance profile to prednisolone, vincristine and L-asparaginase (PVA) has prognostic significance in childhood de novo ALL (Den Boer JCO 2003, Styczynski JCO 2004). So far, no data exist to support prognostic value of in vitro drug resistance profile in childhood acute myeloid leukemia (AML). The aim of this study was the analysis of prognostic value of ex vivo drug resistance profile in childhood AML. A total number of 90 children (46 male, 44 female) aged 0.1–17 yrs (median 10 yrs), with de novo AML were included into the study. All patients were recruited between 1999 and 2004 and were treated according to ANLL-98 protocol. Patients with early deaths were excluded from the study. Drug resistance profile was done by the MTT assay in one laboratory for 4–24 drugs. According to median cytotoxicity for each of tested drugs, all patients were scored as resistant or sensitive to this drug. Patients who received a HSC transplantation were censored at the time of transplantation. Drug resistance profile and other known prognostic parameters, were examined by Kaplan-Meier method and by multivariate analysis using the Cox regression proportional hazard model. Probability of overall survival was 0.57±0.05, p(LFS)=0.72±0.05, mean LFS 3.36 yrs (95%CI=2.95–3.77). Relapses occurred in 17/90 children; 40/90 children died during follow-up. Children who relapsed during follow-up showed median higher in vitro resistance of leukemic blasts to most of tested drugs, except for cytarabine, cladribine, vincristine, mercaptopurine and thioguanine. Better LFS was observed for patients with favourable cytogenetics (1.00 vs 0.74±0.09, p=0.055); early BM response by day 15 (0.76±0.07 vs 0.54±0.13, p=0.013); BM remission by day 28 of induction (0.74±0.07 vs 0.35±0.26, p=0.018); ex-vivo sensitive patients to cyclophosphamide (0.83±0.15 vs 0.65±0.09, p=0.12), doxorubicin (0.81±0.12 vs 0.64±0.09, p=0.18), epirubicin (0.72±0.13 vs 0.61±0.12, p=0.26), fludarabine (0.73±0.12 vs 0.62±0.11, p=0.22), mitoxantrone (0.77±0.12 vs 0.51±0.13, p=0.07), treosulphan (0.88±0.12 vs 0.62±0.11, p=0.29), etoposide (0.70±0.13 vs 0.63±0.09, p=0.4) and for patients with leukemic cells being sensitive to fludarabine, treosulphan and mitoxantrone ie. FTM score (0.73±0.12 vs 0.50±0.14, p=0.034). In multivariate analysis, two factors showed prognostic value: early BM response by day 15 (p=0.0021; HR=0.29, 95%CI=0.13–0.64) and combined ex vivo drug resistance profile to fludarabine, treosulphan and mitoxantrone (FTM score), p=0.0048; HR=0.38, 95%CI=0.19–0.77. Combined ex vivo drug resistance profile to fludarabine, treosulphan and mitoxantrone has prognostic significance in childhood acute myeloid leukaemia, however cytogenetics and early bone marrow response to therapy seems to have stronger prognostic value.

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